Synthesis of biotin conjugates of the antifungal compound cymoxanil

Article abstract of DOI:10.1002/ps.469

Biotin conjugates are of considerable value in investigating the mode of action of biologically active compounds. Two biotin conjugates related to the antifungal compound cymoxanil [1-(2-cyano-2-methoximinoacetyl)-3-ethyl urea] were prepared as the first step in an effort to employ display cloning to identify the compound's target site. In the first conjugate, prepared in five steps, the biotin moiety was attached at the position occupied by the ethyl group in cymoxanil. In the second conjugate, prepared in four steps, the biotin moiety was attached through the oxime functional group.

Full text of DOI:10.1002/ps.469

Pest Management Science
Pest Manag Sci 58:392±396 ꢀonline: 2002)
DOI: 10.1002/ps.469
Synthesis of biotin conjugates of the antifungal
compound cymoxanil
Karen Anderson Evans,1 Charles T Kane2 and Colin M Tice1*
1Rohm and Haas Co, PO Box 904, 727 Norristown Road, Spring House, PA 19477-0904, USA
2Starks Associates, Inc, 1280 Niagara Street, Buffalo, NY 14213, USA
Abstract: Biotin conjugates are of considerable value in investigating the mode of action of biologically
active compounds. Two biotin conjugates related to the antifungal compound cymoxanil [1- 2-cyano-
2-methoximinoacetyl)-3-ethyl urea] were prepared as the ®rst step in an effort to employ display
cloning to identify the compound's target site. In the ®rst conjugate, prepared in ®ve steps, the biotin
moiety was attached at the position occupied by the ethyl group in cymoxanil. In the second conjugate,
prepared in four steps, the biotin moiety was attached through the oxime functional group.
# 2002 Society of Chemical Industry
Keywords: cymoxanil; biotin conjugate; fungicide
1
INTRODUCTION
suggested two compounds as initial synthetic targets:
conjugate 3, in which the biotin tag isappended to the
ethylamino group, and conjugate 4, in which the biotin
tag isattached through the oxyimino group and which
lacksthe ethylaminocarbonyl group. Thisreport
describes the synthesis of these.
The agricultural antifungal compound cymoxanil
[1-ꢀ2-cyano-2-methoxyiminoacetyl)-3-ethylurea; Fig
1, 1)1 has highly desirable curative activity against a
number of oomycete fungi including Phytophthora
infestans ꢀMont) de Bary, the causative agent of potato
late blight. The value of the curative activity of 1 is
tempered by itslack of residuality, and, asa result, it is
always mixed with other fungicides. The target site of 1
has been suggested to lie in the metabolism of uridine
2
EXPERIMENTAL
2.1 General
2
or itsincorporation into RNA; however, effectson
1H NMR spectra were obtained at 300MHz,
400MHz or 500MHz using Bruker DRX-300, Varian
INOVA 400 or Bruker DRX-500 instrumentation,
respectively. 13C NMR spectra were acquired on a
Bruker DPX300 at 75MHz. IR spectra were obtained
using either Bio-Rad FT540 or Perkin Elmer 1760
instrumentation. Low resolution mass spectra were
obtained using Fisons Platform Quadrupole instru-
mentation for ESI or VG 70-SE instrumentation for
FAB ionization techniques.
other processes, including respiration and amino acid
biosynthesis, have also been noted.3 In order to
identify its target site using display cloning4 of a
Phytophthora infestans cDNA library, we required a
biotin-tagged conjugate of 1.
The strength of the interaction between biotin ꢀ2a,
Fig 1) and avidin or streptavidin has made biotin
conjugatesof both ms all moleculesand biomacro-
moleculesextremely valuable biochemical tools. 4±9 In
the design of bioconjugates, careful consideration
must always be given to the site of attachment of the
conjugated molecule, so that binding to the target
2.2 Syntheses
2.2.1 2-Cyano-2-ꢀmethoxyimino)acetamide ꢀ6)
Dimethyl sulfate ꢀ2.80ml, 29.6mmol) was added to a
mixture of 2-cyano-2-ꢀhydroxyimino)acetamide ꢀ5,
prepared asin Reference 13; 3.00g, 26.5mmol),
potassium carbonate ꢀ4.40g, 31.8mmol), and N,N-
dimethylformamide ꢀ27ml) at 5°C, and the mixture
wasstirred at room temperature for 4h. The mixture
wasthen poured into water ꢀ175ml) and extracted
with ethyl acetate ꢀ2Â175ml). The organic layer was
washed with brine ꢀ150ml), dried over anhydrous
magnesium sulfate, and concentrated to give an off-
white solid. The crude product was recrystallized from
a mixture of ethyl acetate and hexanesto give 2.70g
protein isnot dirsupted. In the caes of
particularly problematic since the structure±activity
1 thisis
10±13
range israther narrow
and it wasunclear where
the biotin tag might be appended without preventing
binding to the target site. Furthermore, a published
study has suggested that an unidenti®ed breakdown
2
product of 1 isresponsible for itsantifungal activity;
however, the structure of the active component has not
been elucidated. In the face of these uncertainties, it
seemed prudent to prepare more than one conjugate.
Analysis of the results of published and in-house
structure±activity studies of compounds related to 1
* Correspondence to: Colin M Tice, RheoGene Inc, PO Box 949, 727 Norristown Road, Spring House, PA 19477-0949, USA
E-mail: ctice@rheogene.com
# 2002 Society of Chemical Industry. Pest Manag Sci 1526±498X/2002/$30.00
392

Synthesis of biotin conjugates of cymoxanil
Figure 1. Cymoxanil, biotin and the conjugates prepared.
ꢀ80%) of 6 asan off-white oslid, 1H NMR ꢀhexa-
deuterodimethyl sulfoxide, 400MHz): d 8.00 ꢀs, 1H),
7.92 ꢀs, 1H), 4.17 ꢀs, 3H).
0°C wastsirred for 0.5h,
p-toluenesulfonic acid
monohydrate ꢀ267mg, 1.40mmol) wasthen added
and the mixture wasstirred at room temperature for
15min. The reaction mixture wasconcentrated to
dryness, co-evaporated with dichloromethane ꢀ10ml),
and the resulting colorless oil was triturated with tert-
butyl methyl ether ꢀ50ml) at room temperature for
1.0h. The suspension was centrifuged and the solvent
was decanted. The solid pellet was washed with diethyl
ether ꢀ20ml) and dried under vacuum at room
temperature to give 595mg ꢀ99%) of ꢀ10) asa white
solid, 1H NMR ꢀhexadeuterodimethyl sulfoxide,
300MHz): d 8.40±8.00 ꢀbm, 3H), 7.98 ꢀt, 1H,
J =5.7Hz), 7.48 ꢀd, 2H, J =8.0Hz), 7.11 ꢀd, 2H,
J =7.8Hz), 4.26 ꢀs, 3H), 3.17 ꢀdd, 2H, J =6.7,
12.8Hz), 2.77 ꢀt, 2H, J =7.5Hz), 2.29 ꢀs, 3H), 1.50
ꢀm, 2H), 1.30 ꢀm, 2H).
2.2.2 N-[5-[[2-cyano-2-ꢀmethoxyimino)-1-
oxoethyl]amino]carbonyl]-N-tert-butoxycarbonyl-1,5-
diaminopentane ꢀ9)
A mixture of 6 ꢀ750mg, 5.90mmol), oxalyl chloride
ꢀ650ml, 7.45mmol) and 1,2-dichloroethane ꢀ17ml)
wasstirred under re¯ux for 1.0h. The reaction mixture
was concentrated under reduced pressure to give
crude 2-cyano-2-ꢀmethoxyimino)acetyl isocyanate
ꢀ7) as a light yellow solid. The solid was treated with
a solution of N-tert-butoxycarbonyl-1,5-diaminopen-
tane ꢀ8, 1.26g, 6.25mmol) in 1,4-dioxane ꢀ20ml), and
the mixture wasstirred at room temperature for 1.5h.
The reaction mixture wasloaded on to a column of
silica gel ꢀ190g, 4cmÂ40cm), packed in hexanesand
eluted with ethyl acetate ‡ hexanesꢀ1 litre of 1 ‡1 by
volume, then 3‡2). Product-containing fractions
were concentrated to give 1.1g ꢀ52%) of 9 asa white
solid, 1H NMR ꢀdeuterochloroform, 400MHz): d
8.41 ꢀs, 1H), 7.90 ꢀm, 1H), 4.51 ꢀm, 1H), 4.31 ꢀs,
3H), 3.31 ꢀdd, 2H, J =7.0, 13.2Hz), 3.10 ꢀdd, 2H,
J =6.6, 13.2Hz) 1.59±1.49 ꢀm, 4H), 1.42 ꢀs, 9H),
1.37±1.33 ꢀm, 2H).
2.2.4 ꢀ3aS, 4S, 6aR)-N-[5-[[[[2-cyano-2-ꢀmethoxy-
imino)-1-oxoethyl]amino]carbonyl]amino]pentyl]-hexa-
hydro-2-oxo-1H-thieno[3,4-d]imidazole-4-pentanamide
ꢀ3)
To a suspension of 10 ꢀ495mg, 1.16mmol) and N-
hydroxysuccinimidobiotin ꢀ396mg, 1.16mmol) in
N,N-dimethylformamide ꢀ5.5ml) at 0±5°C wasadded
triethylamine ꢀ396ml, 2.84mmol). The reaction ¯ask
waswrapped in aluminum foil, and the mixture was
stirred at room temperature for 20h. The resulting
thick, white suspension was diluted with ethyl acetate
ꢀ40ml) and centrifuged. The solvent was decanted,
and the solid pellet was washed with ethyl acetate
ꢀ4Â20ml) and dried to give the crude product
2.2.3 N-[5-[[2-cyano-2-ꢀmethoxyimino)-1-oxoethyl]-
amino]carbonyl]-1,5-diaminopentane p-toluenesulfonate
salt ꢀ10)
A solution of 9 ꢀ500mg, 1.41mmol) in dichloro-
methane ꢀ2.5ml) and tri¯uoroacetic acid ꢀ2.5ml) at
Pest Manag Sci 58:392±396 ꢀonline: 2002)
393

KA Evans, CT Kane, CM Tice
ꢀ463mg). The solid was puri®ed by trituration with
saturated sodium hydrogen carbonate ꢀ2Â20ml),
water ꢀ3Â20ml) and acetonitrile ꢀ3Â20ml). The
resulting solid was dried under vacuum at room
temperature to give 3 ꢀ175mg, 31%) asa white solid;
mp 138±142°C ꢀwith prior softening); 1H NMR
ꢀhexadeuterodimethyl sulfoxide, 500MHz): d 10.3
ꢀs, 1H), 7.92 ꢀt, 1H, J =5.5Hz), 7.70 ꢀt, 1H, J =
5.3Hz), 6.37 ꢀs, 1H), 6.32 ꢀs, 1H), 4.30 ꢀm, 1H), 4.26
ꢀs, 3H), 4.13 ꢀm, 1H), 3.16 ꢀdd, 2H, J =6.6, 12.8Hz),
3.10 ꢀm, 1H), 3.03 ꢀdd, 2H, J =6.5, 12.7Hz), 2.82
ꢀdd, 1H, J =5.1, 12.4Hz), 2.58 ꢀd, 1H, J = 12.4Hz),
2.04 ꢀt, 2H, J =7.4Hz), 1.63±1.25 ꢀm, 12H); 13C
NMR ꢀhexadeuterodimethyl sulfoxide, 75MHz): d
171.8, 162.7, 158.5, 151.5, 126.8, 108.7, 66.1, 61.1,
69.2, 55.5, 38.3, 35.2, 28.9, 28.8, 28.2, 28.1, 25.4,
23.7; two 13C resonances of 3 are obscured by DMSO-
d6 signals; MS ꢀESI) m/z 480 ꢀM-H); IR ꢀpotassium
bromide): 3295, 2935, 2862, 1703, 1641, 1545, 1494,
847mg, 7.49mmol) in tetrahydrofuran ꢀ33ml) was
added dropwise to sodium hydride ꢀ297mg,
7.43mmol, 60% dispersion in mineral oil) at 20°C
under an argon atmosphere. The suspension was
cooled to 0±5°C, and a solution of 12 ꢀ2.20g,
6.26mmol) in tetrahydrofuran ꢀ22ml) wasadded
dropwise. When the addition was complete, the
reaction mixture washeated at re¯ux for 9h and then
stirred at 20°C for 16h. The reaction mixture was
diluted with ethyl acetate ꢀ200ml), washed with water
ꢀ4Â50ml), dried ꢀmagnesium sulfate), and concen-
trated to give the crude product ꢀ3.1g). The crude
material waspuri®ed by column chromatography
ꢀ230g of silica gel eluted with ethyl acetate followed
by ethyl acetate ‡methanol, 98‡2 by volume, until
product began to elute, then ethyl acetate ‡methanol,
95‡5 by volume) to give 13 ꢀ1.9g, 79%), asa light
1
yellow oil, H NMR ꢀhexadeuterodimethyl sulfoxide,
400MHz): d 8.02 ꢀs, 1H), 7.94 ꢀs, 1H), 7.83 ꢀt, 1H,
J =5.1Hz), 6.77 ꢀt, 1H, J =4.2Hz), 4.42 ꢀt, 2H,
J =6.6Hz), 3.03 ꢀdd, 2H, J =6.6, 12.8Hz), 2.90 ꢀdd,
2H, J =6.6, 13.2Hz), 2.18 ꢀt, 2H, J =7.0Hz), 1.97 ꢀt,
2H, J =7.0Hz), 1.39 ꢀs, 9H), 1.39±1.34 ꢀm, 4H),
1.27±1.22 ꢀm, 2H).
1460, 1264, 1241, 1149, 1069, 867, 762, 609cmÀ1
.
No syn/anti determinations were performed for
compoundswith an alkoxyimine moiety; however,
1H and 13C NMR spectra of the 3 and 4 indicate that
they are single isomers and, by analogy with com-
poundsprepared by ims ilar methodsꢀReferences
11±13), they are assumed to possess the E con®gura-
tion.
2.2.7 N-[5-[[4-ꢀ2-Amino-1-cyano-2-oxoethylidene)-
amino]oxy]-1-oxobutyl]-1,5-diaminopentane p-toluene-
sulfonate salt ꢀ14)
2.2.5 N-tert-Butoxycarbonyl-N'-[5-ꢀ4-bromo-1-
oxobutyl)]-1,5-diaminopentane ꢀ12)
A solution of 13 ꢀ907mg, 2.37mmol), in 50%
tri¯uoroacetic acid in dichloromethane ꢀ8.8ml) was
stirred at 0±5°C for 0.5h. p-Toluenesulfonic acid
monohydrate ꢀ450mg, 2.37mmol) wasadded, the
reaction mixture wasstirred at room temperature for
15min. and then concentrated to dryness. The residue
wasco-evaporated with dichloromethane ꢀ3 Â10ml)
then the residue was triturated with tert-butyl methyl
ether ꢀ75ml) at room temperature for 0.5h. The
solvent was decanted from the oily solid residue, which
was dissolved in ethanol ꢀ5ml) and diluted with diethyl
ether ꢀ75ml). The resulting suspension was stirred at
room temperature for 0.5h and then stored at À10°C
for 0.5h. The solvent was decanted and the solid was
dried under vacuum at room temperature to give 14
ꢀ1.05g, 97%), asa white oslidi®ed foam, 1H NMR
ꢀhexadeuterodimethyl sulfoxide, 400MHz): d 8.02 ꢀs,
1H), 7.96 ꢀs, 1H), 7.85 ꢀt, 1H, J =5.9Hz), 7.62 ꢀbs,
3H), 7.49 ꢀd, 2H, J =8.1Hz), 7.13 ꢀd, 2H,
J =8.1Hz), 4.44 ꢀt, 2H, J =6.2Hz), 3.07±3.03 ꢀdd,
2H J =6.6, 12.5Hz), 2.79±2.77 ꢀm, 2H), 2.31 ꢀs, 3H),
2.20 ꢀt, 2H, J =7.0Hz), 2.01±1.95 ꢀm, 2H), 1.58±
1.50 ꢀm, 2H), 1.46±1.39 ꢀm, 2H), 1.34±1.30 ꢀm, 2H).
To
a solution of 4-bromobutyric acid ꢀ2.49g,
14.9mmol) in tetrahydrofuran ꢀ55ml) at À3°C
ꢀice±sodium chloride bath) under an argon atmos-
phere wasadded dropwise 1-methylpiperidine ꢀ1.48g,
14.9mmol) followed by isobutyl chloroformate
ꢀ1.93g, 14.9mmol). The reaction mixture wasstirred
at À3°C for 15min., then 1-methylpiperidine ꢀ1.48g,
14.9mmol) wasadded dropwies, followed by the
dropwise addition of a solution of N-tert-butoxycarbo-
nyl-1,5-diaminopentane ꢀ2.50g, 12.4mmol) in tetra-
hydrofuran ꢀ20ml). The reaction mixture wasstirred
at 0° to À3°C for 3h, then ®ltered and the ®lter cake
washed with tetrahydrofuran ꢀ3Â25ml). The com-
bined ®ltrate and washings were concentrated to
dryness to give a light yellow oil. The oil was puri®ed
by column chromatography ꢀ280g of silica gel eluted
with ethyl acetate ‡hexanes, 3‡1 by volume) to give
12 ꢀ2.55g, 49%) as a viscous, colorless oil that
solidi®ed on standing at À20°C to give a white solid,
1H NMR ꢀdeuterochloroform, 500MHz): d 5.63 ꢀbm,
1H), 4.55 ꢀbm, 1H), 3.48 ꢀt, 2H, J =6.3Hz), 3.25
ꢀdd, 2H, J =6.8, 12.9Hz), 3.11 ꢀbd, 2H, J =6.0Hz),
2.35 ꢀt, 2H, J =7.0Hz), 2.22±2.17 ꢀm, 2H), 1.62±
1.47 ꢀm, 4H), 1.44 ꢀs, 9H), 1.38±1.32 ꢀm, 2H); MS
2.2.8 ꢀ3aS,4S,6aR)-N-[5-[[4-[[ꢀ2-amino-1-cyano-2-
oxoethylidene)amino]oxy]-1-oxobutyl]amino]pentyl]-
hexahydro-2-oxo-1H-thieno[3,4-d]imidazole-4-
pentanamide ꢀ4)
‡
‡
ꢀESI) m/z 351 ꢀM‡H) , 373 ꢀM‡Na) .
2.2.6 N-tert-Butoxycarbonyl-N'-[5-[[4-ꢀ2- amino-1-
cyano-2-oxoethylidene)amino]oxy]-1-oxobutyl]-1,5-
diaminopentane ꢀ13)
To a suspension of 14 ꢀ613mg, 1.35mmol) and N-
hydroxysuccinimidobiotin ꢀ460mg, 1.35mmol) in
N,N-dimethylformamide ꢀ6.4ml) at 5°C wasadded
triethylamine ꢀ460ml, 3.30mmol). The reaction ¯ask
A solution of 2-cyano-2-ꢀhydroxyimino)acetamide ꢀ5,
394
Pest Manag Sci 58:392±396 ꢀonline: 2002)

Synthesis of biotin conjugates of cymoxanil
Figure 2. Synthesis of conjugate 3. (a) Me2SO4, K2CO3, DMF, 5°C → rt, 4h; (b) (COCl)2, ClCH2CH2Cl, reflux, 1h; (c) BocNH(CH2)5NH2 (8), dioxane, rt, 1.5h; (d)
CF3CO2H, CH2Cl2 (1:1), 0.5h; then TsOH.H2O; (e) biotin-NHS (2c), Et3N, DMF, 5°C → rt, 20h.
waswrapped in aluminum foil, and the mixture was
stirred at room temperature for 21h. The reaction
mixture ꢀyellow solution) was added dropwise to
vigorously stirred ethyl acetate ꢀ200ml), the resulting
suspension was stirred at room temperature for 0.5h
and then chilled to 0°C for 1.0h. The gel-like
suspension was centrifuged, the solvent was decanted,
and the solid was washed with ethyl acetate
ꢀ2Â20ml). The solid was dried under vacuum at
room temperature to give the crude product ꢀ716mg).
The solid was puri®ed by trituration with saturated
sodium hydrogen carbonate ꢀ3Â20ml) and water
ꢀ3Â25ml), then dissolved in acetonitrile ꢀ20ml),
®ltered, and concentrated to a residue. The residue
wasco-evaporated with acetonitrile ꢀ3 Â10ml) then
dried under vacuum at room temperature for 6h to
give 4 ꢀ259mg, 38%) asan off-white solid, mp 185±
spectrum ꢀFAB) of 4 shows the expected molecular
ion aswell asa higher massion at m/z 584, which may
correspond to the reaction of glycerol with the terminal
amide of 4 during FAB sample preparation to give the
glyceryl ester analog of 4 with loss of ammonia; IR
ꢀpotassium bromide): n:3299, 3082, 2940, 2862,
1700, 1648, 1547, 1461, 1438, 1266, 1041,
686cmÀ1
;
Calcd for C22H35N7O5SÁ0.5H2O:C,
51.02; H, 6.97; N, 18.81; S, 6.28. Found:C, 51.00;
H, 7.09; N, 18.74; S, 6.15.
No syn/anti determinations were performed for
compoundswith an alkoxyimine moiety; however,
1H and 13C NMR spectra of the 3 and 4 indicate that
they are single isomers and, by analogy with com-
poundsprepared by ims ilar methodsꢀReferences
11±13), they are assumed to possess the E con®gura-
tion.
1
187°C ꢀdec) ꢀuncorrected); H NMR ꢀhexadeutero-
dimethyl sulfoxide, 400MHz): d 8.02 ꢀs, 1H), 7.95 ꢀs,
1H), 7.84 ꢀt, 1H, J =5.1Hz), 7.74 ꢀt, 1H, J =5.5Hz),
6.43 ꢀs, 1H), 6.37 ꢀs, 1H), 4.43 ꢀt, 2H, J =6.2Hz),
4.34±4.31 ꢀm, 1H), 4.16±4.13 ꢀm, 1H) 3.14±3.09 ꢀm,
1H), 3.06±3.00 ꢀm, 4H), 2.86±2.82 ꢀdd, 2H, J =5.1,
2.5Hz), 2.59 ꢀd, 1H, J =12.1Hz), 2.19 ꢀt, 2H,
J =7.0Hz), 2.06 ꢀt, 2H, J =7.3Hz), 2.01±1.94 ꢀm,
2H), 1.68±1.25 ꢀm, 12H); 13C NMR ꢀhexadeutero-
dimethyl sulfoxide, 75MHz): d 172.9, 171.0, 162.8,
159.0, 127.5, 108.5, 77.5, 61.1, 59.2, 55.5, 38.5,
32.3, 35.2, 31.2, 28.9, 28.8, 28.3, 28.1, 25.4, 24.6,
24.9; one 13C resonance of 4 isobscured by DMSO- d6
3
DISCUSSION
The known acylisocyanate 7 appeared to be a suitable
intermediate for the synthesis of 3 ꢀFig 2) and was
prepared in two steps from the amide 5 following the
literature procedure.13 Our initial plan wasto react 7
with biotin derivative 2b to afford 3 directly. Some
similar reactions have been reported in the literature,7
however, in our handsreaction of 7 and 2b failed in
both DMF and dioxane at room temperature, possibly
due to the poor solubility of 2b in both these solvents.
In an attempt to overcome the apparently poor
solubility of 2b, it washeated with 7 at 80°C in
‡
signals; MS ꢀFAB) m/z 510 ꢀM‡H) ; the mass
Figure 3. Synthesis of conjugate 4. (a) 1-Methylpiperidine, i-BuOCOCl, THF, 0°C; (b) H2NCOC(CN)=NOH (5), NaH, THF, 0°C → reflux, 9h; (c) CF3CO2H,
CH2Cl2 (1:1), 0.5h; then TsOH.H2O; (d) biotin-NHS (2c), Et3N, DMF, 5°C → rt, 21h.
Pest Manag Sci 58:392±396 ꢀonline: 2002)
395

KA Evans, CT Kane, CM Tice
2 Leroux P, Fritz R and Despreaux D, The mode of action of
cymoxanil, in Botrytis cinerea, in Pesticide science and biotechnol-
ogy, ed by Greenhalgh R and RobertsTR, Blackwell, Oxford,
pp 191±196 ꢀ1987).
DMF, but 7 appeared to decompose without forming
3 under these conditions. A three-step procedure from
7 to 3 proved successful. Thus, isocyanate 7 reacted
smoothly with 8, the mono-Boc derivative of 1,5-
diaminopentane, to afford urea 9 in acceptable yield.
De-protection of 9 with tri¯uoroacetic acid in di-
chloromethane gave the amine 10, which wasisolated
as its tosylate salt. Coupling of 10 with biotin
N-hydroxysuccinimide ester ꢀ2c) provided conjugate
3.
3 ZiogasBN and Davidse LC, Studiesof the mechanism of action
of cymoxanil in Phytophthora infestans. Pestic Biochem Physiol
29:89±96 ꢀ1987).
4 Sche PP, McKenzie KM, White JD and Austin DJ, Display
cloning: functional identi®cation of natural product receptors
using cDNA-phage display. Chem Biol 6:707±716 ꢀ1999).
5 Hermanson GT, Bioconjugate techniques, Academic Press, San
Diego, pp 570±592 ꢀ1996).
The synthesis of conjugate 4 ishsown in Fig 3.
Coupling of 8 with 4-bromobutanoic acid ꢀ11) using
either DCC or EDC ꢀwater-soluble carbodiimide)
failed to give 12 in our hands; however, preparation of
the mixed anhydride of 11 with isobutyl chloro-
formate, followed by reaction with 8 afforded 12.
O-Alkylation of 5 with 12, using sodium hydride as
base, led to the desired oxime 13. The Boc protecting
group wasremoved from 13 with tri¯uoroacetic acid in
dichloromethane and the resulting amine 14 was
isolated as its tosylate salt. Amine 14 wasreacted with
2c to afford conjugate 4. In conclusion, we have
described the synthesis of two conjugates related to the
antifungal-compound 1-ꢀ2-cyano-2-methoxyimino-
acetyl)-3-ethylurea ꢀcymoxanil, 1). The application
of these conjugates in biological studies will be
reported separately.
6 Janda KD, Lo LC, Lo CHL, Sim MM, Wang R, Wong CH and
Lerner RA, Chemical selection for catalysis in combinatorial
antibody libraries. Science ꢀWashington) 275:945±948 ꢀ1997).
7 Konoki K, Sugiyama N, Murata M and Tachibana K, Direct
observation of binding between biotinylated okadaic acids and
protein phosphatase 2A monitored by surface plasmon
resonance, Tetrahedron Lett 40:887±890 ꢀ1999).
8 Adam GC, Cravatt BF and Sorensen EJ, Pro®ling the speci®c
reactivity of the proteome with non-directed activity-based
probes, Chem Biol 8:81±95 ꢀ2001).
9 Hashimoto M, Yang J and Holman GD, Cell-surface recognition
of biotinylated membrane proteinsrequiresvery long ps acer
arms: an example from glucose-transporter probes. Chem Bio
Chem 1:52±59 ꢀ2001).
10 Alexandri AA, Baicu T and Safta M, Oxime derivativesactivity.
I. In vitro biological activity. An Inst Cercet Prot Plant Acad
Stiinte Agric Silvice 18:165±171 ꢀ1985).
11 Smith PH, Chamberlain K, SugarsJM and Bromilow RH,
Fungicidal activity of N-ꢀ2-cyano-2-methoxyiminoacetyl)-
amino acidsand their derivative.s
ꢀ1995).
Pestic Sci 44:219±224
ACKNOWLEDGEMENTS
The authorsare indebted to DrsGlenn R Carlson and
David A Starksfor their support.
12 Smith PH, Chamberlain K, SugarsJM and Bromilow RH,
Fungicidal activity of N-ꢀ2-cyano-2-methoxyiminoacetyl)-
methionine and itsderivativesand analogue.s
Pestic Sci
45:357±361 ꢀ1995).
13 Kai H, Nakayama K, Tsuji H, Masuko M and Takase A,
Synthesis and fungicidal activity of 2-alkoxyimino-2-cyano-
acetamide derivatives. Nihon Noyaku Gakkaishi ꢀJ Pestic Sci)
23:44±48 ꢀ1998).
REFERENCES
1 Davidson SH, 2-Cyano-2-hydroxyiminoacetamides as plant
disease control agents, US Patent 3 954 992 ꢀ1976).
396
Pest Manag Sci 58:392±396 ꢀonline: 2002)